Portal de Periódicos da CAPES

Collapsin Response Mediator Proteins of Neonatal Rat Brain Interact with Chondroitin Sulfate

2003; Elsevier BV; Volume: 278; Issue: 5 Linguagem: Inglês

10.1074/jbc.m210181200

1083-351X

Sebastian Franken, Ulrich Junghans, Volker Rosslenbroich, Stephan L. Baader, Ralf Hoffmann, Volkmar Gieselmann, Christoph Viebahn, Joachim Kappler,

Hippo pathway signaling and YAP/TAZ

Chondroitin sulfate proteoglycans are structurally and functionally important components of the extracellular matrix of the central nervous system. Their expression in the developing mammalian brain is precisely regulated, and cell culture experiments implicate these proteoglycans in the control of cell adhesion, neuron migration, neurite formation, neuronal polarization, and neuron survival. Here, we report that a monoclonal antibody against chondroitin sulfate-binding proteins from neonatal rat brain recognizes collapsin response mediator protein-4 (CRMP-4), which belongs to a family of proteins involved in collapsin/semaphorin 3A signaling. Soluble CRMPs from neonatal rat brain bound to chondroitin sulfate affinity columns, and CRMP-specific antisera co-precipitated chondroitin sulfate. Moreover, chondroitin sulfate and CRMP-4 were found to be localized immuno-histochemically in overlapping distributions in the marginal zone and the subplate of the cerebral cortex. CRMPs are released to culture supernatants of NTera-2 precursor cells and of neocortical neurons after cell death, and CRMP-4 is strongly expressed in the upper cortical plate of neonatal rat where cell death is abundant. Therefore, naturally occurring cell death is a plausible mechanism that targets CRMPs to the extracellular matrix at certain stages of development. In summary, our data indicate that CRMPs, in addition to their role as cytosolic signal transduction molecules, may subserve as yet unknown functions in the developing brain as ligands of the extracellular matrix. Chondroitin sulfate proteoglycans are structurally and functionally important components of the extracellular matrix of the central nervous system. Their expression in the developing mammalian brain is precisely regulated, and cell culture experiments implicate these proteoglycans in the control of cell adhesion, neuron migration, neurite formation, neuronal polarization, and neuron survival. Here, we report that a monoclonal antibody against chondroitin sulfate-binding proteins from neonatal rat brain recognizes collapsin response mediator protein-4 (CRMP-4), which belongs to a family of proteins involved in collapsin/semaphorin 3A signaling. Soluble CRMPs from neonatal rat brain bound to chondroitin sulfate affinity columns, and CRMP-specific antisera co-precipitated chondroitin sulfate. Moreover, chondroitin sulfate and CRMP-4 were found to be localized immuno-histochemically in overlapping distributions in the marginal zone and the subplate of the cerebral cortex. CRMPs are released to culture supernatants of NTera-2 precursor cells and of neocortical neurons after cell death, and CRMP-4 is strongly expressed in the upper cortical plate of neonatal rat where cell death is abundant. Therefore, naturally occurring cell death is a plausible mechanism that targets CRMPs to the extracellular matrix at certain stages of development. In summary, our data indicate that CRMPs, in addition to their role as cytosolic signal transduction molecules, may subserve as yet unknown functions in the developing brain as ligands of the extracellular matrix. extracellular matrix chondroitin sulfate collapsin response mediator protein CS proteoglycan fetal calf serum phosphate-buffered saline monoclonal antibody electrospray ionization-mass spectrometry guanidinium hydrochloride 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid matrix-assisted laser desorption ionization time-of-flight spectroscopy The extracellular matrix (ECM)1 of the developing brain has a unique composition from lecticans, tenascins, laminins, and hyaluronic acid (1Ruoslahti E. Glycobiology. 1996; 6: 489-492Google Scholar, 2Luckenbill-Edds L. Brain Res. Brain Res. Rev. 1997; 23: 1-27Google Scholar, 3Rauch U. Cell Tissue Res. 1997; 290: 349-356Google Scholar, 4Margolis R.U. Margolis R.K. Cell Tissue Res. 1997; 290: 343-348Google Scholar, 5Lander A.D. Curr. Opin. Neurobiol. 1993; 3: 716-723Google Scholar). Lecticans are proteoglycans that carry chondroitin sulfate (CS) side chains on core proteins encompassing a N-terminal hyaluronan binding domain and a C-terminal lectin domain. Four different members of the lectican protein family are known (neurocan, aggrecan, versican, and brevican) (1Ruoslahti E. Glycobiology. 1996; 6: 489-492Google Scholar). Their multidomain composition enables lecticans to interact with multiple cell surface molecules and diffusible ligands (see below) (6Bandtlow C.E. Zimmermann D.R. Physiol. Rev. 2000; 80: 1267-1290Google Scholar,7Rauch U. Feng K. Zhou X.H. Cell. Mol. Life Sci. 2001; 58: 1842-1856Google Scholar). Chondroitin sulfates are glycosaminoglycans composed of repeated glucuronic acid-[β1,3]-N-acetylgalactosamine disaccharide units that are linked by [β1,4]-glycosidic bonds into long unbranched chains with molecular masses of up to 50 kDa and more (8Kjellen L. Lindahl U. Annu. Rev. Biochem. 1991; 60: 443-475Google Scholar). With respect to the position of sulfate esters at the galactosamine, CSA (C-4-S, containing C-4-sulfate) and CSC (C-6-S, containing C-6-sulfate) are distinguished. The expression of these proteoglycan core proteins is precisely tuned during brain development (9Miller B. Sheppard A.M. Bicknese A.R. Pearlman A.L. J. Comp. Neurol. 1995; 355: 615-628Google Scholar). Moreover, the disaccharide composition of CS is regulated (9Miller B. Sheppard A.M. Bicknese A.R. Pearlman A.L. J. Comp. Neurol. 1995; 355: 615-628Google Scholar,10Margolis R.K. Rauch U. Maurel P. Margolis R.U. Perspect. Dev. Neurobiol. 1996; 3: 273-290Google Scholar). The functions of chondroitin sulfate proteoglycans (CS-PGs) in neural tissue can be categorized into effects on cell adhesion, cell migration, neurite formation, neuron polarization, synaptic modulation/plasticity, and neuron survival (for reviews, see Ref. 6Bandtlow C.E. Zimmermann D.R. Physiol. Rev. 2000; 80: 1267-1290Google Scholar and references therein and Ref. 11Kappler J. Junghans U. Koops A. Stichel C.C. Hausser H.J. Kresse H. Müller H.W. Eur. J. Neurosci. 1997; 9: 306-318Google Scholar). These effects often critically depend on glycosaminoglycans or may even be attributed solely to glycosaminoglycan chains. Many of the listed functions, however, have been discovered using in vitro experiments that were designed in a way that the proteoglycan or glycosaminoglycan component became limiting in the assays. In vivo, on the other hand, there is marked structural redundancy of ECM components (1Ruoslahti E. Glycobiology. 1996; 6: 489-492Google Scholar, 3Rauch U. Cell Tissue Res. 1997; 290: 349-356Google Scholar) if one considers for example the existence of four different lecticans (see above). Thus, phenotypes of knockout animals lacking a single ECM protein are often rather mild (for example in knockouts for neurocan (12Zhou X.H. Brakebusch C. Matthies H. Oohashi T. Hirsch E. Moser M. Krug M. Seidenbecher C.I. Boeckers T.M. Rauch U. Buettner R. Gundelfinger E.D. Fässler R. Mol. Cell. Biol. 2001; 21: 5970-5978Google Scholar) and tenascin-C (13Forsberg E. Hirsch E. Frohlich L. Meyer M. Ekblom P. Aszodi A. Werner S. Fässler R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6594-6599Google Scholar, 14Fukamauchi F. Mataga N. Wang Y.J. Sato S. Youshiki A. Kusakabe M. Biochem. Biophys. Res. Commun. 1996; 221: 151-156Google Scholar)). Inactivation of enzymes involved in CS biosynthesis, on the other hand, could lead to more severe phenotypes since CS is a component of multiple ECM proteins. In the chondroitin-6-sulfate transferase knockout mouse, however, no major CNS pathology was found (15Uchimura K. Kadomatsu K. Nishimura H. Muramatsu H. Nakamura E. Kurosawa N. Habuchi O., El- Fasakhany F.M. Yoshikai Y. Muramatsu T. J. Biol. Chem. 2002; 277: 1443-1450Google Scholar). C-6-S may be replaced by C-4-S, and expression data suggest that C-4-S is probably more important in the developing brain. Nevertheless, the importance of CS in vivois underscored impressively by the observation that after local treatment with chondroitinase ABC, which degrades C-4-S, C-6-S, and dermatan sulfate, regeneration of functional neurites in the adult spinal cord is enabled (16Bradbury E.J. Moon L.D. Popat R.J. King V.R. Bennett G.S. Patel P.N. Fawcett J.W. McMahon S.B. Nature. 2002; 416: 636-640Google Scholar). Thus, CS-PGs are considered to contribute to the inhibition of regenerative responses in the adult mammalian nervous system. The current insight into the mechanisms of how CS acts on neurons is still rudimentary. Binding partners of CS-PGs at the plasma membrane include sulfatide and several (eventually glycosylphosphatidylinositol-anchored) cell adhesion molecules of the Ig family such as N-CAM, L1, TAG-1, and F3/contactin (for a review, see Ref. 6Bandtlow C.E. Zimmermann D.R. Physiol. Rev. 2000; 80: 1267-1290Google Scholar). The signaling events exerted after binding of CS-PGs to these molecules are unknown. On the other hand, CS-PGs bind a variety of soluble ligands including growth factors like bovine fibroblast growth factor and oligomeric glycoproteins of the ECM-like tenascins (6Bandtlow C.E. Zimmermann D.R. Physiol. Rev. 2000; 80: 1267-1290Google Scholar). Interestingly, CS is a critical component of a molecular scaffold to which diffusible molecules are bound that convey inhibitory or promoting actions, e.g. on the adhesion of thalamic neurons and the formation of neurites (17Emerling D.E. Lander A.D. Neuron. 1996; 17: 1089-1100Google Scholar). The identity of these diffusible molecules, however, is as yet unknown. To elucidate the molecular basis of the neurotrophic actions of chondroitin sulfate, we previously fractionated protein extracts from neonatal rat brain on a chondroitin sulfate affinity column and used the eluted binding proteins to generate monoclonal antibodies (18Junghans U. Franken S. Pommer A. Müller W. Viebahn C. Kappler J. Histochem. Cell Biol. 2002; 117: 317-325Google Scholar). One of these antibodies, termed mAb-9, recognizes a 65-kDa protein with laminar expression in the neocortex, which parallels the expression of CS. The protein is present in both the fraction of soluble proteins and in the particulate fraction of neonatal rat brain. The aim of the present study was to identify this chondroitin sulfate-binding protein and to characterize its interaction with glycosaminoglycans. We show that mAb-9 recognizes a soluble protein that is present in the cytosol, termed collapsin response mediator protein-4 (CRMP-4). This protein and its relatives interact with chondroitin sulfate, and they are released from the cytosol of neurons to the extracellular space most probably after cell death. This may explain why CRMP-4 was found to be co-localized with chondroitin sulfate in the developing neocortex of rat brain in regions where naturally occurring cell death is prevalent. Unless otherwise stated, chemicals were from Serva (Heidelberg, Germany), Sigma, Roche Molecular Biochemicals, or Merck. ZERO Blunt vector for PCR cloning was from Invitrogen, and pQE-30 vector for bacterial expression of histidine-tagged proteins was from Qiagen (Hilden, Germany). SDS-PAGE was performed on 5–15% gradient slab gels (Bio-Rad Protean II) or 10% mini-gels (Bio-Rad MiniProtean) (19Laemmli U.K. Nature. 1970; 227: 680-685Google Scholar). Gels were stained by silver (20Ansorge W. J. Biochem. Biophys. Methods. 1985; 11: 13-20Google Scholar), Coomassie Blue, or zinc/imidazole (21Fernandez-Patron C. Calero M. Collazo P.R. Garcia J.R. Madrazo J. Musacchio A. Soriano F. Estrada R. Frank R. Castellanos-Serra L.R. Mendez E. Anal. Biochem. 1995; 224: 203-211Google Scholar). For Western blotting, proteins were transferred to nitrocellulose in a semi-dry blotting apparatus (Bio-Rad) according to Kyhse-Andersen (22Kyhse-Andersen J. J. Biochem. Biophys. Methods. 1984; 10: 203-209Google Scholar). After blocking with 3% nonfat dry milk powder, 1% bovine serum albumin in Tris-buffered saline containing 0.05% Tween 20 (TBST), the first antibody incubation was performed for 1 h at room temperature in TBST. Bound antibodies were visualized after binding of peroxidase-labeled secondary antibodies with enhanced chemiluminescence (ECL kit, AmershamBiosciences). Whole brains from neonatal Wistar rats were shock-frozen in liquid nitrogen and homogenized in a Dounce homogenizer in 10 mm HEPES, pH 7.4, 2 mmMgCl2 (HEPES buffer) containing 2 mm Pefabloc, 1 mm leupeptin, and 1 mm pepstatin. The homogenate was centrifuged at 10,000 × g for 15 min, and the supernatant was subsequently centrifuged at 100,000 ×g for 1 h to obtain soluble protein. Forty milligrams of this protein material were loaded on a 1-ml Mono Q fast protein liquid chromatography column (Amersham Biosciences), washed with 10 column volumes of loading buffer (HEPES buffer), and eluted stepwise with 5 column volumes of HEPES buffer containing 100, 300, 500, and 2000 mm NaCl. One-milliliter fractions were collected and analyzed by SDS-PAGE, silver staining and Western blot with mAb-9. Western positive fractions were concentrated (Centricon10, Millipore) and loaded on a 25-ml Superose-12 column (Amersham Biosciences). Again, 1-ml fractions were collected and analyzed by Western blotting. For mass spectrometry, proteins were prepared according to the method of Gevaert et al. (23Gevaert K. Verschelde J.L. Puype M. Van Damme J. Goethals M., De Boeck S. Vandekerckhove J. Electrophoresis. 1996; 17: 918-924Google Scholar). Briefly, the positive fractions were concentrated by ultrafiltration and separated by SDS-PAGE. After staining with zinc/imidazole the protein bands corresponding to the Western signal of the monoclonal antibody were excised. After destaining in 5% citric acid for 15 min and another 3 times for 15 min in deionized water and incubation in SDS-PAGE sample buffer containing 0.1% SDS, 10% glycerol, 50 mm dithiothreitol, 12 mm Tris/HCl, pH 6.8, and 0.1% bromphenol blue for 1 h, the gel was cut into small pieces (5 × 5 mm) and loaded on top of a concentration gel (5% acrylamide, 0.26% bisacrylamide, 125 mm Tris/HCl, pH 6.8, and 0.1% SDS) inside of a Pasteur pipette (length, 145 mm). The pipette was transferred to an isoelectro-focusing unit (Bio-Rad), and the gel pieces were carefully overlayered with running buffer (50 mm Tris, 190 mm glycine, 0.1% SDS). Electrophoresis at 250 V was continued until the bromphenol blue approached the lower edge of the pipette. The gel was removed from the Pasteur pipette and stained with Coomassie Blue, and the sharp blue protein band in the lower part of the pipette was excised, destained, and stored at −20 °C until further analysis. Aliquots of this material were analyzed by Western blotting to confirm hat the desired protein had been excised. The destained gel piece was washed twice in digestion buffer (10 mmNH4HCO3) for 15 min and twice for 15 min in digestion buffer/acetonitrile 1:1 (v/v). The gel piece was re-swollen with 2 μl of protease solution (trypsin at 0.05 μg/μl in digestion buffer), and after 20 min another 10 μl of digestion buffer was added. After digestion overnight at 37 °C the supernatant was collected and dried down to about 0.5 μl. For nanospray ESI-MS 2 μl of 70% formic acid were added, and this solution was used in 0.5-μl aliquots. ESI-MS was done as described (24Immler D. Gremm D. Kirsch D. Spengler B. Presek P. Meyer H.E. Electrophoresis. 1998; 19: 1015-1023Google Scholar) using a TSQ 7000 Triple quadrupole mass spectrometer (Finnigan MAT, San Jose, CA) equipped with the standard ESI source or an in-house constructed nanospray source. The ESI-voltage was between 0.6 and 1.1 kV for nanospray. Mass spectra were acquired with a scan speed of 1000 Da/s. For ESI-MS/MS analysis, argon at a pressure of 3 millitorr was used as collision gas. Data acquisition and evaluation were done on a DEC work station using the ICIS software, version 8.2.1. Peptide mass calculation was done with the BIOWORKS software, version 8.2.1. Peptide masses obtained from in-gel digestion were used for searching the SwissProt.r34 data base with MS-FIT (falcon.ludwig.ucl.ac.uk/msfit.htm). The standard parameters were: species Rattus norvegicus, molecular mass 40–100 kDa, tryptic digest with a maximum of 1 missed cleavage site. Peptide masses were assumed to be monoisotopic, and cysteine was assumed to be not modified. The allowed mass error was set at 0.1%. First-strand cDNA was amplified after reverse transcription of 3 μg of a mixture of E17/P4 rat brain RNA using Superscript reverse transcriptase (Invitrogen) according to the manufacturer's instructions using an oligo-dT primer. After digestion with RNase H one-tenth of the reaction was used as template for PCR amplification by Pfu polymerase (Stratagene, La Jolla, CA) using 94 °C for 90 s, 55 °C for 45 s, 72 °C for 2 min for 30 cycles. The following primer sequences were used: CRMP-1(5′), 5′-ATGTCTCATCAGGGGAAGAAGAG-3′, CRMP-1(3′), 5′-TATCTGGCGCATCTGAGGTCAACC-3′, CRMP-2(5′), 5′-ATGTCTTATCAGGGGAAGAAAAAT-3′, CRMP-2(3′), 5′-GCAGGCCTAGGAGCTTTAGCCCAG-3′), CRMP-3(5′), 5′-ATGTCCTTCCAAGGCAAGAAGAGCATTCCCCGGATA-3′, CRMP-3(3′), 5′-TGCCAGACCCCAAGTCTAAGAAAG-3′, CRMP-4(5′), 5′-CAGAATCGCCACCATGTC-3′, CRMP-4(3′), 5′-GAGGGCTTAACTCAGGGA-3′. The PCR products were cloned into the ZERO Blunt vector and sequenced using an ABI Prism 310 Genetic Analyzer. Bacterial overexpression inEscherichia coli was performed using the Qiaexpress kit (Qiagen) according to the instructions of the manufacturer. The coding regions of the four CRMP-cDNAs were re-amplified using primers containing restriction sites for SalI and HindIII and the pCR Blunt vectors containing the CRMP inserts as templates. The following primer sequences were used: CRMP-1(5′)Sal, 5′-ACCTAGCGTCGACACATAGAAGGTAGAATGTCTCATCAGGGG-3′; CRMP-1(3′)Hind, 5′-CGCCGCAAGCTTTATCTGGCGCATCTGAGG-3′; CRMP-2(5′)Sal, 5′-ACCTAGCGTCGACACATAGAAGGTAGAATGTCTTATCAGGGG-3′; CRMP-2(3′)Hind, 5′-CGCCGCAAGCTTGCAGGCCTAGGAGCTTTA-3′; CRMP-3(5′)Sal, 5′-ACCTAGCGTCGACACATAGAAGGTAGAATGTCCTTCCAAGGC-3′; CRMP-3(3′)Hind, 5′-CGCCGCAAGCTTTGCCAGACCCCAAGTCTA-3′; CRMP-4(5′)Sal, 5′-ACCTAGCGTCGACACATGTCCTACCAGGGCAAGAAG-3′; CRMP-4(3′)Hind, 5′-CGCCGCAAGCTTACTCAGGGATGTGATGTTAGA-3′. The PCR products were gel-purified, digested with SalI and HindIII, and ligated into the pQE-30 expression vector, thereby incorporating a 5′ extension of the cDNA coding for a His6 tag. M15[pRep] bacteria were transformed, and ampicillin/kanamycin resistant strains were analyzed for CRMP cDNA inserts and sequenced to verify ligation sites and the PCR products. Positive strains were grown in Luria Bertani medium containing kanamycin and ampicillin, and expression was induced by 2 mmisopropyl-1-thio-β-d-galactopyranoside. Cells were lysed in Tris/phosphate buffer containing 8 m urea, and expression of recombinant His-tagged CRMP proteins was tested by Western blot with an anti-RGS-His antibody (Qiagen). Antibodies raised in rabbit against the synthetic peptides CRMP-4-pep (FDLTTTPKGGTPAGSTRGSPTRPN, rCRMP-4 residues 504–527) and CRMP-Fam-pep (SFYADIYMEDGLIKQIGDN, rCRMP-4 residues 30–48) were obtained from Pineda Antiköper Service, Berlin, Germany. CRMP-1-pep (YEVPATPKHAAPAPSAKSSPSKHQ, rCRMP-1 residues 504–527), CRMP-2-pep-a (CEVSVTPKTVTPASSAKTSPAKQQ, rCRMP-2 residues 504–527), CRMP-2-pep-b (GIQEEMEALVKDHGV, rCRMP-2 residues 147–161), CRMP-3-pep (HEVMLPAKPGSGTQARASCSGKIS, rCRMP-3 residues 496–519) were synthesized as described (25Kappler J. Franken S. Junghans U. Hoffmann R. Linke T. Müller H.W. Koch K.W. Biochem. Biophys. Res. Commun. 2000; 271: 287-291Google Scholar) and coupled to keyhole limpet hemocyanin as described (26Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 59-123Google Scholar) via additional N-terminal cysteine residues. The peptide-keyhole limpet hemocyanin conjugates were used to immunize New Zealand White rabbits (Lammers, Euskirchen, Germany). For the first immunization 200 μg of peptide dissolved in Freund's complete adjuvant (Sigma) was injected subcutaneously. Each animal was boosted twice at intervals of 4 weeks with the same amount of antigen in incomplete Freund's adjuvant (Sigma). For the immunization protocol, special permission according to Section 8 of the German Law on the Protection of Animals had been obtained from the Bezirksregierung Köln. All rabbit antisera were used at a dilution of 1:10,000 for Western blotting. Monospecific IgG was purified by immunoaffinity chromatography with the peptides immobilized on thiol-Sepharose (Amersham Biosciences) as described (26Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 59-123Google Scholar). Purified IgG was ultra-filtered into 50 mm NaHCO3, pH 8.5. After the addition of 10 μl of 150 mm NHS-Biotin (Pierce) the antibody solution was incubated at 4 °C overnight. IgG was then separated from the unreacted free biotin by size exclusion chromatography on NAP10 columns (Amersham Biosciences). Chondroitin sulfate was coupled to EAH-Sepharose (Amersham Biosciences) as described (18Junghans U. Franken S. Pommer A. Müller W. Viebahn C. Kappler J. Histochem. Cell Biol. 2002; 117: 317-325Google Scholar). As control columns, heparin Hitrap, SP Hitrap, and CM Hitrap 1-ml columns (Amersham Biosciences) were used. Approximately 10 mg of soluble neonatal rat brain proteins obtained after ultracentrifugation of postnuclear supernatants at 100,000 × g for 1 h were filtered (0.45 μm) and chromatographed on 1-ml analytical columns using an Äkta Explorer equipment (Amersham Biosciences). Runs on CS columns and control columns were carried out in parallel, taking advantage of the column-scouting routine of the Unicorn 3.1 software using sequential step elution with PBS containing 300 mm NaCl, 750 mm NaCl, 2 m NaCl, and 4 mguanidinium hydrochloride (GuaHCl) as described (18Junghans U. Franken S. Pommer A. Müller W. Viebahn C. Kappler J. Histochem. Cell Biol. 2002; 117: 317-325Google Scholar). To remove the salt from the eluent fractions and to concentrate the proteins, they were precipitated with acetone (−20 °C) and washed twice with 80% ethanol (4 °C) before Western blot analysis with the peptide-specific antibodies against CRMPs. 1-ml aliquots of soluble neonatal rat brain proteins prepared as described above were incubated with 1 μl of the different peptide-specific CRMP antisera for 1 h at 4 °C and with 10 μl of protein A-agarose (Calbiochem) for an additional hour at 4 °C with continuous rocking. The precipitates were collected by centrifugation, washed 3 times in phosphate-buffered saline, dissolved in 100 μl of Laemmli buffer, and analyzed by Western blotting with the biotinylated CRMP-Fam antibody or CS56 (Sigma) against CS. Before brain dissection, animals were perfused with standard mammalian Ringer's solution, pH 7.4, followed by 3.7% formaldehyde. Brains were post-fixed for 16 h and washed extensively in tap water. After dehydration in a series of increasing ethanol concentrations, brain tissue was embedded in paraffin, and 10-μm sections were cut on a microtome (Leica HM 355 S). Sections were mounted on Histobond slides, dried for 2 days at 37 °C, and used for immunohistochemistry. First, sections were deparaffinized and hydrated by decreasing concentrations of ethanol in H2O. Afterward, sections were incubated in boiling 2× SSC (1× SSC = 0.15 m NaCl and 0.015 m sodium citrate) for 20 min. After equilibrating in PBS, sections were treated with 1% H2O2 in PBS for 10 min to remove endogenous peroxidase activity. After permeabilization with 0.5% Triton X-100 in PBS for 10 min, sections were washed with PBS and incubated in a 4% bovine serum albumin, PBS solution for at least 30 min. First, antibodies to CRMP-4 were used in a dilution of 1:1000, and for CS56 (Sigma), in a dilution of 1:200 (all in 4% bovine serum albumin, PBS). Sections were incubated at 4 °C overnight. After washing, sections were treated with the secondary biotinylated antibody diluted 1:200 in 4% bovine serum albumin, PBS, washed again, and incubated in a streptavidin-peroxidase complex (ABC-kit, Vector). After a 1-h incubation, sections were washed intensively and stained with diaminobenzidine (0.05% in Tris-buffered saline). Counterstaining was done with hemalum (Mayers hemalum, 1:6 dilution in H2O, Merck) for 5 min followed by several washes in H2O and a final wash in tap water. Pictures were taken with a digital camera (Polaroid DMC Ie, Cambridge, UK) connected to a Zeiss Axioskop 2 (Jena). NTera-2 precursor cells (Stratagene) were grown in Dulbecco's modified Eagle's medium/F-12 supplemented with 10% fetal calf serum (FCS), l-glutamine, and penicillin/streptomycin. Cell death was induced by feeding the cells with medium without FCS after repeated washings with serum-free medium. Conditioned medium was harvested after 3 days and centrifuged at 1000 × g for 15 min to remove floating cells and debris. For Western blot analysis, conditioned medium was subjected to Q-Sepharose (Hitrap 1-ml column, Amersham Biosciences) chromatography to capture CRMPs using elution with a linear gradient from 150 mm to 1 m NaCl in phosphate buffer. Primary neocortical neurons were prepared as described (27Junghans U. Koops A. Westmeyer A. Kappler J. Meyer H.E. Müller H.W. Eur. J. Neurosci. 1995; 7: 2341-2350Google Scholar) and plated onto poly-d-lysine-coated 10-cm cell culture Petri dishes (Falcon) or 6-well cell culture plates (Sarstedt). Cultures were incubated at 37 °C in humidified 10% CO2, 90% air for 16–24 h and analyzed by phase contrast microscopy. To visualize the morphology of nuclei, cultures were fixed for 10 min in 4% paraformaldehyde, washed with PBS, and stained briefly with 4,6-diamidine-2-phenylindole (100 μg/ml in PBS). Pictures were taken with a digital camera (Axiovision, Zeiss) connected to a Zeiss Axiovert 100M, and images were processed with the Axiovision software (Zeiss, Göttingen, Germany). Protein determination was performed with the Bradford assay or the detergent-compatible protein assay (both purchased from Bio-Rad) using bovine serum albumin as the standard. Lactate dehydrogenase activity was determined as described (28Storrie B. Madden E.A. Methods Enzymol. 1990; 182: 203-225Google Scholar). The monoclonal antibody mAb-9 used in this study recognizes a 65-kDa protein that is abundant in the soluble fraction of neonatal rat brain (18Junghans U. Franken S. Pommer A. Müller W. Viebahn C. Kappler J. Histochem. Cell Biol. 2002; 117: 317-325Google Scholar). From this material the 65-kDa protein was captured on a Mono Q anion exchange column (Fig. 1). When steps of increasing ionic strength were applied to the column, about 30% of the cross-reacting protein was eluted with 200 mm sodium chloride, and the remaining 70% was released at 500 mmsodium chloride according to Western blot analysis (Fig. 1 C). Interestingly, the electrophoretic mobility of the cross-reacting protein bands from the eluate fractions was slower in comparison to the starting material (Fig. 1C). Because the 200 mm NaCl eluate contained a lower amount of contaminating proteins than the 500 mm eluate, according to silver-stained SDS-gels (Fig. 1 B), fraction 7 from the Mono Q column was subsequently fractionated using size exclusion chromatography on a Superose 12 column (Fig. 2). From this column the cross-reacting protein eluted after 11–12 ml, corresponding to a molecular mass of ∼200 kDa (Fig. 2, A and C). Finally, purification to homogeneity was achieved by preparative SDS-PAGE (data not shown). In-gel digestion with trypsin yielded 22 peptides, which were analyzed by ESI-MS. In the SwissProt.r34 rat sequence data base (Table I) 10/22 peptide masses fitted to a rat sequence homologous to “Turned on after division” protein of 64 kDa (TOAD-64 (29Minturn J.E. Fryer H.J. Geschwind D.H. Hockfield S. J. Neurosci. 1995; 15: 6757-6766Google Scholar), also called collapsin response mediator protein-2 (CRMP-2 (30Goshima Y. Nakamura F. Strittmatter P. Strittmatter S.M. Nature. 1995; 376: 509-514Google Scholar)). Extension of the search to the entire mammalian data base (nrdb, EMBL Heidelberg), however, showed a more close match of 15/22 peptides to mouse CRMP-4 (mUlip, Unc33-like phosphoprotein (31Byk T. Dobransky T. Cifuentes-Diaz C. Sobel A. J. Neurosci. 1996; 16: 688-701Google Scholar)). Furthermore, MS/MS analyses of three different peptides from the spectrum confirmed that these were derived from the rat homologue of CRMP-4. Because only a truncated cDNA sequence for rat CRMP-4 was available in the data base, a chimeric sequence was assembled from the available truncated rat CRMP-4 and mouse CRMP-4/mUlip. To this chimeric sequence 19 of the 22 obtained masses could be matched exactly (data not shown). Thus, the mass spectrometric analyses indicate that mAb-9 recognizes collapsin response mediator protein-4 from rat brain.Figure 2Gel filtration chromatography. Fraction 7 from the Mono Q chromatography was subjected to gel filtration chromatography on a Superose 12 column. A, chromatogram. Antigen containing fractions are marked with a bar, and the positions of the molecular weight standards (in kDa) is indicated byarrows. Silver staining of SDS-PAGE (B) and Western blot (C) with the mAb-9. Bars indicate the position of apparent molecular weight marker bands (in kDa).View Large Image Figure ViewerDownload (PPT)Table IMS-Fit search results of the peptide masses obtained after digestion of the unknown protein, recognized by mAb-9 with trypsinData submittedM+H matchedΔ %StartEndPeptide sequenceModifications766.8000766.45750.0447558565(R)IVAPPGGR(A)878.6000878.44460.0177362368(R)MSVIWDK(A)1010.00001010.6111−0.0605488496(R)LAELRGVPR(G)1016.0001015.55360.0440259268(K)SAAEVIAGAR(K)1310.40001310.6897−0.02216475(R)MVIPGGIDVHTR(B)IMet-ox1642.40001643.7892−0.08